The subject of plant protection against pathogens remains the area of utmost importance in agriculture. Many commercially valuable agricultural crops are prone to infection by plant viruses and fungi capable of inflicting significant damage to a crop in a given season, and drastically reducing its economic value. The reduction in economic value to the farmer in turn results in a higher cost of goods to ultimate purchasers. Several published studies have been directed to the expression of plant virus capsid proteins in a plant in an effort to confer resistance to viruses. See, e.g., Abel et al., Science 232:738-43 (1986); Cuozzo et al., Bio/Technology 6:549-57 (1988); Hemenway et al., EMBO J. 7:1273-80 (1988); Stark et al., Bio/Technology 7:1257-62 (1989); and Lawson et al., Bio/Technology 8:127-34 (1990). However, the transgenic plants exhibited resistance only to the homologous virus and related viruses, but not to unrelated viruses. Kawchuk et al., Mol. Plant-Microbe Interactions 3(5):301-07 (1990), disclose the expression of wild-type potato leafroll virus (PLRV) coat protein gene in potato plants. Even though the infected plants exhibited resistance to PLRV, all of the transgenic plants that were inoculated with PLRV became infected with the virus and thus disadvantageously allowed for the continued transmission of the virus such that high levels of resistance could not be expected. See U.S. Pat. No. 5,304,730.
Lodge et al., Proc. Natl. Acad. Sci. USA 90:7089-93 (1993), report the Agrobacterium tumefaciens-mediated transformation of tobacco with a cDNA encoding wild-type pokeweed antiviral protein (PAP) and the resistance of the transgenic tobacco plants to unrelated viruses. PAP, a Type I ribosome-inhibiting protein (RIP) found in the cell walls of Phytolacca americans (pokeweed), is a single polypeptide chain that catalytically removes a specific adenine residue from a highly conserved stem-loop structure in the 28S rRNA of eukaryotic ribosomes, and interferes with elongation factor-2 binding and blocking cellular protein synthesis. See, e.g., Irvin et al., Pharmac. Ther. 55:279-302 (1992); Endo et al., Biophys. Res. Comm., 15:1032-36 (1988); and Hartley et al., FEBS Lett. 290:65-68 (1991). The observations by Lodge were in sharp contrast to previous studies, supra, which reported that transgenic plants expressing a viral gene were resistant to that virus and closely related viruses only. See also Beachy et al., Ann. Rev. Phytopathol. 2:451-74 (1990); and Golemboski et al., Proc. Natl. Acad. Sci. USA 87:6311-15 (1990). Lodge also reports, however, that the PAP-expressing tobacco plants (i.e., above 10 ng/mg protein) tended to have a stunted, mottled phenotype, and that other transgenic tobacco plants that accumulated the highest levels of PAP were sterile. RIPs have proven unpredictable in other respects such as target specificity. Unlike PAP which (as demonstrated in Lodge, supra), ricin isolated from castor bean seed is 1000 times more active on mammalian ribosomes than plant ribosomes. See, e.g., Harley et al., Proc. Natl. Acad. Sci. USA 79:5935-5938 (1982). Barley endosperm RIP also shows very little activity against plant ribosomes. See, e.g., Endo et al., Biochem. Biophys. Acta 994:224-226 (1988) and Taylor et al., Plant J. 5:827-835 (1984).
Fungal pathogens contribute significantly to the most severe pathogen outbreaks in plants. Plants have developed a natural defense system, including morphological modifications in their cell walls, and synthesis of various anti-pathogenic compounds. See, e.g., Boller et al., Plant Physiol 74:442-444 (1984); Bowles, Annu. Rev. Biochem. 52:873-907 (1990); Joosten et al., Plant Physiol. 82:945-951 (1989); Legrand et al., Proc. Natl. Acad. Sci. USA 84:6750-6754 (1987); and Roby et al., Plant Cell 2:999-1007 (1990). Several pathogenesis-related (PR) proteins have been shown to have anti-fungal properties and are induced following pathogen infection. These are different forms of hydrolytic enzymes, such as chitinases and .beta.-1,3-glucanases that inhibit fungal growth in v by destroying fungal cell walls. See, e.g., Boller et al., supra; Grenier et al., Plant Physiol. :1277-123 (1993); Leah et al., J. Biol. Chem. 266:1464-1573 (1991); Mauch et al., Plant Physiol. 87:325-333 (1988); and Sela-Buurlage Buurlage et al., Plant Physiol. 101:857-863 (1993).
Several attempts have been made to enhance the pathogen resistance of plants via recombinant methodologies using genes encoding pathogenesis-related proteins (such as chitinases and .beta.-1,3-glucanases) with distinct lytic activities against fungal cell walls. See, e.g., Broglie et al., Science 254:1194-1197 (1991); Vierheilig et al., Mol. Plant-Microbe Interact. 6:261-264 (1993); and Zhu et al., Bio/Technology 12:807-812 (1994). Recently, two other classes of genes have been shown to have potential in conferring disease resistance in plants. Wu et al., Plant Cell 1:1357-1368 (1995), report that transgenic potato expressing the Aspergillus niger glucose oxidase gene exhibited increased resistance to Erwinia carotovora and Phytophthora infestans. The hypothesis is that the glucose oxidase-catalyzed oxidation of glucose produces hydrogen peroxide, which when accumulates in plant tissues, leads to the accumulation of active oxygen species, which in turn, triggers production of various anti-pathogen and anti-fungal mechanisms such as phytoalexins (see Apostol et al., Plant Physiol. 20:109-116 (1989) and Degousee, Plant Physiol. 945-952 (1994)), pathogenesis-related proteins (Klessig et al., Plant Mol. Biol. 26:1439-1458 (1994)), strengthening of the plant cell wall (Brisson et al., Plant Cell 6:1703-1712 (1994)), induction of systemic acquired resistance by salicylic acid (Chen et al., Science 162:1883-1886 (1993)), and hypersensitive defense response (Levine et al., Cell 79:583-593 (1994)).
In addition to the studies on virus resistance in plants, RIPs have been studied in conjunction with fungal resistance. For example, Logeman et al., Bio/Technology 10:305-308 (1992), report that a RIP isolated from barley endosperm provided protection against fungal infection to transgenic tobacco plants. The combination of barley endosperm RIP and barley class-II chitinase has provided synergistic enhancement of resistance to Rhizoctonia solani in tobacco, both in v and in vivo. See, e.g., Lea et al., supra; Mauch et al., supra; Zhu et al., supra; and Jach et al., The Plant Journal 8:97-109 (1995). PAP, however, has not shown antifungal activity in vitro. See Chen et al., Plant Pathol. 40:612-620 (1991), which reports that PAP has no effect on the growth of the fungi Phytophthora infestans, Colletotrichum coccodes, fusarium solani, fusarium sulphureum, Phoma foreata and Rhizoctonia solani, in vitro.
Hence, a need remains for a means by which to confer broad spectrum virus and/or fungus resistance to plants without causing cell death or sterility, and which requires a minimum number of transgenes.